This invention relates to the use of a naturally occurring sugar-phosphate compound called fructose-1,6-diphosphate, for treating the sporadic crises that arise in people suffering from sickle cell anemia.
The following paragraphs provide background information on sickle cell anemia, under its subheading, and then on fructose-1,6-diphosphate, under a different subheading. However, it must be emphasized that fructose-1,6-diphosphate (abbreviated herein as FDP) apparently has never before been used to treat sickle cell anemia. A search of the National -2O Library of Medicine computerized database, combining "sickle cell anemia" or "sickle hemoglobin" with either "fructose diphosphate" or "fructose phosphates" identified only a single article, which involved a different compound. That article, Colomer et al 1991 (complete citations are provided below) related to the levels of fructose-2,6-bisphosphate in certain types of cells, congenital hemolytic anemias. As described below, 2,6-FDP (which is of no interest whatever in the current invention) has very different biochemical properties than 1,6-FDP, which is the compound used in this invention. The 1,6-FDP isomer (with phosphate groups coupled to the #1 and #6 carbon atoms of the fructose molecule) is the only form of FDP that is of interest herein. It is discussed in more detail below.
Accordingly, sickle cell anemia and 1,6-FDP have both been studied extensively. However, there apparently has never been any prior effort to treat sickle cell anemia, using 1,6FDP.
Background Information on Sickle Cell Anemia Sickle cell anemia is a well-known disease, in which red blood cells (abbreviated as RBC's; also called erythrocytes) contain hemoglobin molecules which have a dangerous tendency to crystallize and become non-functional, due to a genetic mutation. In the most common form of this disease, the beta (or B) chain of the hemoglobin protein has a valine residue, instead of a glutamate residue, at the number 6 position. Other substitutions have also been identified, such as "sickle C" disease (which has a lysine residue at the #6 position) and "sickle D" disease (which has a glutamine residue at the #6 residue).
This genetic mutation is relatively common in Africa, since a person who carries a single copy of the mutated gene has a relatively high resistance to malaria, without suffering from major adverse health effects. Accordingly, it has been estimated that roughly 30% of all people native to Nigeria (as just one example) carry at least one such gene (Barnhart et al 1976). About 8% of African-Americans also carry at least one such gene, although local populations often contain higher levels. The gene which disposes red blood cells to sickling is often referred to as HbS, where "Hb" refers to hemoglobin, and "S" refers to sickling. By contrast, a normal, healthy, adult hemoglobin is usually referred to as HbA.
As briefly noted above, a single copy of the gene does not inflict major adverse health effects on the person carrying that gene. Such people are often referred to as "heterozygotes," since they carry two different types of genes (i.e., one is the defective HbS gene, and the other is the normal HbA gene). Usually, only about 40% or less of their hemoglobin is of the sickling variety, while the rest (the majority) is normal. Their red cells will sickle, but only if exposed to hypoxia at much more severe levels than will provoke sickling in homozygotes. Heterozygotes (single-gene carriers) usually have few if any clinical problems, and they do not suffer from reduced life expectancies or increased hospitalization rates.
By contrast, "homozygotes" carry two copies of the defective HbS gene, and do not have any normal and healthy HbA hemoglobin (they may sometimes retain variable amounts of a fetal type of hemoglobin, called HbF, which does not occur in healthy adults). Accordingly, they are the ones who suffer from the debilitating, often devastating effects of sickle cell anemia. They usually suffer severe spleen damage by the age of about 6, and for the rest of their lives, they suffer from elevated levels of gradually accumulating damage to their other organs and tissues, including the liver and kidneys. In the United States, despite having advanced medical care, typical life expectancy for men having the disease is 42 years, and 48 years for women (Platt et al 1994).
The abnormal amino acid residue on the beta chain allows hemoglobin to polymerize, when it is subjected to a condition of even relatively mild hypoxia. This polymerization activity can be observed in intact red cells, or in cell-free hemoglobin solutions. Polymers are typically 14 stranded helices, and are up to 20 nm long. The formation of polymerized hemoglobin inside red cells provokes a change in the shapes and structures of red blood cells, causing an increase in rigidity of the cell wall, and deformation of the cell into a dehydrated, flattened curved shape, which is the classic "sickle" shape (named after the old grain-harvesting tool) that gives the disease its name. Hemoglobin polymerization can also lead to other severely deformed cell shapes, in addition to the sickled shape. For a review of this molecular process and its effects on red blood cells, see Dean and Schechter 1978.
In a patient, these changes in red blood cell shapes may be widespread (for example, during surgery that requires anesthesia), or regional (for example, behind a venous tourniquet when drawing a blood sample). They can also be triggered by events such as bacterial or viral infections, which stress the body (or certain parts of the body) in various ways that can generate localized ischemia and/or hypoxia.
For most patients, sickle cell anemia does not cause constant or chronic pain. However, most sickle cell anemia patients suffer from sporadic yet recurrent episodes that are referred to herein as "ischemic crises" (all references herein to a crisis or crises refer to these sporadic, recurrent crises which occur in the normal course of sickle cell disease; such references do not relate to any other type of ischemic crisis, such as a stroke or heart attack). During such crises, a sickle cell (SC) patient will usually experience severe pain, at one or more locations which frequently vary between patients, and between different crises in a specific patient. It is not uncommon for one or more joints to become swollen and sore, and/or for the patient to suffer from either sharp or diffuse pain in the abdomen, which is presumed to be due to ischemic conditions in one or more portions of one or more organs.
Such crises are generally referred to as ischemic crises, since they typically involve blockade of capillaries and prevention of blood flow into a tissue that becomes starved of oxygen and glucose. This blockade of the capillaries is caused by red blood cells that have lost their normal shape and flexibility, and have collapsed or distorted into the rigid or semi-rigid "sickled" shapes that gives the disease its name. This blockade of capillaries shuts off the flow of fresh blood through those portions of the organ or tissue that are normally serviced by the blocked capillaries. This state of events, due to the sickling of the red blood cells due to the polymerization of HbS-type hemoglobin molecules, leads directly to ischemia, which is the medical term for inadequate blood flow to an organ or tissue.
Most sickle cell patients usually suffer several such ischemic crises per year. During these crises, the patient usually must be hospitalized, restricted to bed rest with little or no exertion, and treated with a variety of drugs, including strong painkillers such as morphine, codeine, and meperidine (also known as Demerol.TM.), and by broad-spectrum antibiotics, both to help control any infections that may be contributing to the crises, and to help prevent or reduce additional infections in tissues or organs that are weakened by the ischemic crisis.
The physiological damage and increased morbidity and mortality caused by sickle cell anemia has been studied extensively (e.g., Platt et al 1994). Briefly, among young children, dactylitis is common, due to ischemic necrosis of the small bones and cartilages of the hands and feet, and acute abdominal pain is often caused by accumulating damage to the spleen. Acute abdominal pain can also be due to liver or kidney infarction, or associated with hematuria. Cholecystitis due to gall stones, aseptic necrosis of the head of the femur, radiologic evidence of widened marrow space in the skull, spinal osteoporosis and renal papillary necrosis typically occur over the age of 10 years, and pathological fractures often supervene in patients greater than 18 years, especially at the head of the femur and the humerus. Leg ulcers are common in adult patients, and lobar pneumonias, pulmonary infarctions, stroke, and retinal lesions may occur. In all cases, the pain is severe, often migratory, and diffuse.
When an ischemic crisis commences, it sets into motion a cascade of events that render the crisis even more severe and difficult to arrest and treat. Three aspects of the cascading, self-perpetuating nature of these ischemic crises are worth noting.
First, as soon as a single problematic cell takes on a semi-rigid shape and gets wedged into a capillary without being able to pass through it, it can trap a substantial number of cells behind it, in the vasculature which leads toward up to the blocked capillary. The trapped cells eventually become depleted of the oxygen, and they too will begin to sickle and die, which aggravates the size and severity of the problem.
Second, the creation of blockage problems in certain parts of a complex fluid-flow network will quickly transfer additional stresses to the other parts of the network. If a segment of tissue that is normally served by two or more capillaries suddenly loses its supply of fresh nutrients and oxygen from one of its suppliers, it will immediately begin placing higher and greater demands on its other suppliers, thereby placing them in danger of becoming overloaded as well, in a way which can threaten to provoke hypoxic sickling of blood cells in other previously unaffected capillaries, which may then shut down those other capillaries as well.
And third, metabolic rates inside red blood cells increase, when the cells encounter the types of stress that provoke sickling. This increase in metabolism is associated with defective ion transport across the red cell membrane, calcium influx, and increased membrane rigidity. In a sense, when a cell encounters the type of stress that can provoke sickling, its internal mechanisms begin trying to work harder, in order to overcome the problem. However, the increased rate of metabolism can quickly deplete the cell's ATP and other resources, thereby leaving the depleted cell even more vulnerable, and at greater risk of collapsing into a full-blown sickled condition.
The only prior treatment for sickle cell anemia that has shown any substantial benefit involves a compound called hydroxyurea. In some patients, this compound can reduce the frequency of sickle cell crises, presumably due to an ability to increase the expression levels of fetal hemoglobin genes. However, this treatment has only limited utility; many patients on hydroxyurea still experience recurrent ischemic attacks, and it is not effective in treating those attacks. Many of the patients in the clinical trial described in Example 6 were already receiving hydroxyurea therapy, when they suffered the crisis that caused them to enter the study described below, involving FDP.
This is a very brief overview of an extraordinarily difficult, burdensome, and expensive medical problem, which faces many millions of people around the globe, and which imposes huge and intractable financial burdens on sickle cell patients, their families, their employers, their health insurers, and their governmental health, housing, and welfare systems. Under current medical practice, there is a major and severe need for better ways to treat sickle cell anemia, both to prevent the recurrent ischemic crises that characterize the disease, and to help treat the extremely painful ischemic crises that play havoc with the lives (and internal organs) of most sickle cell patients, several times a year. The only care such patients receive today, during such crises, is essentially palliative care, which includes days and days of enforced bed rest, broad-spectrum antibiotics, and pain-killing drugs that need to be potent and effective, but which pose a major risk of long-term addiction. Despite all the advances of modern medical practice, sickle cell anemia remains a severe and debilitating disease, with no effective cure or treatment.
As noted above, there has not been any previously reported effort (to the best of the Applicant's knowledge), by anyone, to use fructose-1,6-diphosphate (FDP) to treat sickle cell anemia, or even to test FDP to determine whether it might be effective in treating sickle cell patients. That point needs to be kept in mind during the next section, which describes the prior art relating to FDP.
Background Information on 1,6-FDP
Fructose-1,6-diphosphate (FDP) is a naturally occurring sugar-phosphate molecule, which is created and then quickly consumed as an intermediate during the series of reactions that make up glycolysis (i.e., the series of reactions by which glucose is metabolized, to release its stored energy). As a short-lived intermediate that is quickly consumed, FDP normally is present in cells only at relatively low concentrations.
It should be noted that some scientists refer to FDP as fructose-1,6-biphosphate, or fructose-1,6-bisphosphate. The 1,6-isomer of fructose diphosphate, which contains phosphate groups bonded to the #1 and #6 carbon atoms of the fructose molecule, is the only isomer of interest herein. Other isomers (such as fructose-2,6-diphosphate) are not relevant herein, and are excluded from any references herein to FDP or fructose diphosphate.
From an energy-containing standpoint, FDP is at the highest point in the pathway of glycolysis; two molecules of energy-rich ATP have to be used, in order to convert the initial glucose molecules into FDP. Starting with FDP, all remaining reactions in the pathway of glycolysis release, rather than consume, energy.
Because of FDP's vantage point at the highest energy plateau of the energy-generating process of glycolysis, which is absolutely essential to all cells, numerous medical and scientific articles have suggested that FDP might potentially be useful as a medical treatment for patients and victims suffering from medical crises such as strokes or brain injury, cardiac arrest, heart attack, suffocation, loss of blood due to injury, shooting, or stabbing, etc. Examples of such articles include Markov et al 1980, 1986, and 1987, Cacioli et al 1988, Lazzarino et al 1989, Crescimanno et al 1990, Myers et al 1990, Gregory et al 1990, Nakai et al 1991, Gobbel et al 1994, Hardin et al 1994, Kelleher et al 1995, and Sano et al 1995. Relevant U.S. patents include U.S. Pat. Nos. 4,546,095 (Markov 1985), 4,703,040 (Markov 1987), and 4,757,052 (Markov 1988).
Despite all of these published articles and patents, which stretch back nearly 20 years, to at least 1980, a high degree of skepticism and reluctance still exists regarding the use of FDP for any medical purpose. Except for a few small and very limited clinical trials, FDP simply is not used or prescribed by any practicing physicians, for any reason or for any medical purpose, except possibly in a few foreign countries such as China and Italy.
The absence of any actual use of FDP on patients (many of whom desperately need the energy supplies that might be derived from FDP, as they are dying of massive heart attacks, cardiac arrest, strokes, or blood loss) is believed to be due to a number of factors, including the following:
(1) FDP is a diphosphate with a strong negative charge, and doctors and researchers widely assume that its strong negative charge will prevent it from entering cells in any significant or useful quantity. Since energy metabolism and glycolysis occur inside cells, it is assumed that FDP will not get to the sites where it is needed, in sufficient quantities to do any significant good.
(2) It is also believed that FDP has a very short half-life in the blood, and will effectively disappear from the blood within a few minutes after injection or infusion. (3) In nearly all types of cells (other than red blood cells, which do not have mitochondria, and which do not engage in aerobic glycolysis), the amount of energy generated during glycolysis (i.e., the conversion of glucose to pyruvic acid) is only a small fraction of the energy generated by the aerobic (Krebs Cycle) oxidation of pyruvic acid, to form carbon dioxide and water. Therefore, under conditions of tissue ischemia or hypoxia, where an oxygen deficit blocks aerobic conversion and causes the creation of lactic acid instead, it is generally assumed that FDP infusion would be insufficient to supplement ATP levels to a degree that can significantly aid cell survival.
(4) Under conditions of ischemia or hypoxia, an injection of FDP into a patient would lead directly to substantial increases in lactic acid levels; for example, when radiolabelled FDP was added to intact isolated hearts that were being perfused, nearly 90% of the exogenous FDP was converted into lactic acid, and only about 10% of the FDP was fully oxidized to carbon dioxide (Lazzarino et al 1992). This effect from an FDP injection could be very harmful, since excess lactic acid can poison an enzyme called phosphofructokinase (PFK), which is a crucial rate-limiting enzyme in glycolysis (e.g., Hoffman 1976; Kubler and Spieckerman 1978; Opie 1968). The potential costs and risks of providing a relatively small quantity of energy (via FDP infusion into ischemic tissue) is very high. If the PFK enzyme is inhibited or poisoned by lactate (which is produced from FDP at a nearly 90% rate, in ischemic tissue), then it is widely assumed that the overall result of FDP administration might well be to inhibit or even shut down the much more useful and productive aerobic (Krebs) pathway which leads to carbon dioxide.
(5) Drug intervention in acute ischemic trauma has proven to be extremely difficult and complex, for a large number of reasons. Among other things, it often requires 1 to 3 hours (or more) before a patient can be properly diagnosed in a manner that justifies the use of a specific drug. This is aggravated by the fact that there are, for example, three major categories of shock, and proper treatment for one type can actually increase the damage suffered by someone who is suffering from a different type of shock. To avoid any liability for improper care, ambulance attendants and other emergency-care providers simply cannot and do not take the risk, in most cases, of trying to treat someone suffering from shock or other medical crises of unknown origin. However, by the time someone arrives at a hospital emergency room and is adequately diagnosed so that focused drug treatment can begin, too much time has often elapsed, and any drugs administered there are often too late to prevent any cell death and permanent tissue damage that has already occurred.
(6) Contrary to the articles cited above which report that FDP may have beneficial effects in certain types of lab tests, a number of other articles have reported that FDP had no beneficial effects in other studies. Examples of these negative articles include Eddy et al 1981, Pasque et al 1984, Tortosa et al 1993, and Angelos et al 1993.
For these and other reasons, it appears that little if any effort has been directed by the pharmaceutical industry toward developing FDP as a useful drug. Under the laws enforced by the U.S. Food and Drug Administration, FDP cannot be sold in the United States for administration to human patients by physicians. With the possible exception of a few small clinical trials, FDP simply is not administered to any patients, anywhere in the United States, regardless of how desperate their plight may be following a stroke, cardiac arrest, shooting, stabbing, or other medical crisis.
More importantly for the subject invention, FDP apparently has never previously been used (or even tested) for treating patients suffering from sickle cell anemia, either for long-term care, or for treating the recurrent ischemic crises caused by sickle cell anemia.
Additional Difficulties in Evaluating Candidate Treatments
There are several additional factors that have severely thwarted further progress in efforts to treat sickle cell anemia. These factors need to be understood and evaluated carefully, in evaluating any such efforts to find ways to overcome these extraordinary difficulties.
First, it should be noted that there are no accepted animal models for studying sickle cell anemia. This makes research on potential treatments for sickle cell anemia substantially more difficult, expensive, and risky. Preliminary tests can be done on an in vitro basis, using red blood cells in blood samples or in cell incubation media. However, if those test results seem satisfactory, there is no middle plateau that allows testing on mice, rats, dogs, or any other type of lab animal. Instead, a giant leap must be made from in vitro tests, all the way up to human clinical trials, which are extremely expensive, liability-laden, and risky. This factor has severely aggravated the lack of more progress toward finding effective treatments.
In addition, the tendency of sickle cell anemia to cause very different types of physiological damage, in different patients, poses even more difficulty in proving that any particular treatment is indeed useful and effective. Damage caused by sickle cell disease can be manifested in nearly any of the internal organs, and the actual pattern of damage that appears in any specific patient is nearly impossible to predict. Furthermore, each type of damage that appears in the various organs is similar to the damage caused by various other diseases and etiologies. In addition, as noted above, the damage tends to accumulate slowly, over a period of decades.
All of these factors, acting together in human patients, make it extraordinarily difficult to develop any treatments that might be able to help ameliorate the symptoms or damage of sickle cell disease, because it is painfully clear, to any pharmaceutical company that might be considering investing in any such research, that it will never be able to obtain approval, from the U.S. Food and Drug Administration or from any of the comparable agencies in other countries, to sell its new drug as an approved therapy for treating sickle cell anemia, unless it can submit trial data which clearly prove that the treatment actually works, in humans. The difficulties that must be overcome to actually prove that a drug can reduce the damage of a disease which takes decades to fully inflict and manifest its damage, and affects different specific patients in very different ways, are extraordinary. These obstacles have severely retarded any progress (or even research) toward effective treatments for sickle cell anemia, and these factors cannot be ignored when evaluating a new treatment, as set forth in the current invention.
However, these confounding factors have been avoided, by the decision of the Applicant company to focus on an entirely different category of treatment outcome, which has never previously been considered significant in any other research to evaluate the effects of FDP. This treatment outcome involves the measurement of pain, which requires patients to answer questions ("How much does it hurt?" ) that require subjective opinions, rather than objective data that can be measured by mechanical devices or chemical reactions.
This newly adopted goal of the planned research raised its own set of questions, doubts, and risks, and it should be noted that this research project was proposed, in detail, in a grant application that was submitted to the National Institutes of Health. That grant application was rejected, by the experts who reviewed that proposal. However, the researchers who were planning the clinical trials described herein deemed this approach to be necessary, as the only practical way to gather useful data that could overcome the difficulties that have confounded other efforts to prove (to the satisfaction of the Food and Drug Administration) the efficacy of candidate treatments for sickle cell disease.
Accordingly, the primary question that was addressed in these clinical trials was the reduction of pain, i.e., the efficacy of FDP as an analgesic drug. This was a highly unusual and non-obvious approach, given the fact that FDP has never previously been reported to offer analgesic (pain-controlling) activity, in any other treatment setting. To the best of the Applicant's knowledge and belief, this is the first-ever disclosure that FDP has analgesic activity, in any situation.
Therefore, one object of the subject invention is to provide a method for reducing pain in patients who suffer from sickle cell anemia, during the recurrent ischemic crises that are attributable to the disease, by disclosing (for the first time) that fructose-1,6-diphosphate (FDP) actually has effective analgesic activity, in such patients, during such crises.
Another object of this invention is to disclose that FDP can reduce the length of hospital stays that are necessary to cope with recurrent sickling crises in sickle cell patients, and can also reduce the need for potentially addictive pain-killers such as morphine and other opiates.
Another object of this invention is to provide a medical treatment for recurrent sickle cell anemia crises, for use in conjunction with other medical treatments that are conventionally used during such crises, in a manner which does not interfere with such other treatments and which reduces pain for the patient and helps facilitate and speed up satisfactory resolution of the crisis, thereby allowing the patient to spend less time in a hospital or other such medical facility.
Another object of this invention is to disclose a method of reducing the accumulative permanent damage that is inflicted on sickle cell patients, by reducing both (1) the amount of damage that organs and tissues suffer due to ischemia caused by blockage of blood flow through capillaries, and (2) the amount of damage caused by oxidative free radicals, in organs and tissues where blood flow has been restored after a period of ischemia.
These and other objects of the invention will become more apparent through the following summary, drawings, and description of the preferred embodiments.